ENVIRONMENTAL

Ex. No. 1.
Objective
.SAMPLING AND PRESERVATION
tudy and understand the
Dreservation techniques for analysis methodologies for collecting a
Introduction
representative sample and
lhe aim of sampling is to
collect representative sample.
which relative proportions or
concentration of all Representative sample by means a sample in
being sampled. The sample volume shall optimal pertinent components will be the same as in the material
analytical purposes. Because of the
small enough that it can be
increasing placed on verifying the transported and large enough for
data, greater emphasissis placed on proper
exercise addresses the collection and
sample collection, tracking, andaccuracy and representatives of
preservation of water, preservation techniques. This
the general principless also apply to the sampling of solids or wastewater and contaminated soil
General Requirements for semisolid matrices. samples;
water/wastewater sampling
Cosure that all sampling
equipment is clean and
Rontainers that are clean and quality-assured before use. Use sample
free of
organic analysis sampling. contaminants. Bake at 450°C all
bottles to be used for
rll sample containers
added nreservative and
without pre-rinsing with sample;
pre-rinsing results in loss of any pre
sides of the container. sometimeson bias results high when certain components adhere to the
can
organic compound Depending or determinations to be performed, fill the
container full (most
determinations)
inorganic analyses). Except when leave space for aeration, mixing, etc.
airspace equivalent to sampling for analysis of volatile organic (microbiological and
during shipment.
approximately 1% of the container volume to allow forcompounds, leave an
thermal exDansion
Special precautions are necessary for
samples containing organic
present at low concentrations compounds orand trace metals.
Since many constituents may be
liter), they may be totally or (micro-grams nanograms per
partially lost or easily contaminated
preservation procedures are not followed. when proper sampling and
For metals it often is
appropriate to collect both a
differentiate between total and dissolved metals presentfiltered and an unfiltered sample to
in the matrix. Be aware that
metals may partially sorb to filters. some
Record of sample shall be as follows
General information
Specific information
"Sample identification number
"Location "Water temperature
" Weather
"Sample collector
" Stream blow
"Date and hour
"Sample type (Grab or "Water level
composite)
ne intormation may be attached tag,
"Any other information
labeling or writing on container with water proof ink.
Description of
By map using sampling points
Use global landmarks
positioning system
Wnen samples are collected from a river, stream, lakes, and
reservoirs observed results may vary
Win seasonal stratification, diurnal variations., rainfall,
runoff, wind depth, stream flow, and
distance from each shore. Selection of the number and distribution of sites at which samples
should be collected depends on study objectives, streamcharacteristics, available equipment, and
other factors. If equipment is available, take an integrated sample from top to bottom in the
 middle of the main channel of the steam or
from side to side at mid-depth. If only
or catch
samples can be collected, preferably take them at various points of equal distance across the
grab
take it in the middle of the main channel of
steam; ifonly one sample can be collected, the stream
andat mid-depth.
Safety Considerations
Always prohibit eating, drinking, or smoking near samples, sampling locations,
and in the
laboratory. Keep sparks, flames, and excessive heat sources away from samples
locations. If lammable compounds are suspected of known to be present and
and sampling
samples are to be
refrigerated, use only specially designed explosion-proof refrigerators.
Label adequately any sample known or suspected to be hazardous
corrosivity, toxicity, oxidizing chemicals, or radioactivity, so that because of flammability,
taken during sample handling, storage, and disposal. appropriate precautions can be
Collection of water/wastewater samples
Types of samples
a. Grab samples (catch samples): Grab samples are single collected at a
specific spot at a site over a short period of time (typically seconds or
mìnutes), Thus, they represent a "snapshot" in both space and time of a
sampling area. Discrete grab samples are taken at a selected location.
depth, and time. Depth integrated grab samples are
collected over a
predetermined part of the entire depth of a water column, at a selected
location and time in a given body ofwater.
Depch sampler or closng botle
Several grab sarnples taken at different tirmes
in the same location.
Weight sildes
Covn cord to
deces

a comterm
ple

Grab sample Depth sampler (grab sample) Composite sample

b. Composite samples: Composite samples should provide a more representative sampling of

heterogeneous matrices in which the concentration of the analytes of interest may vary over short
periods of time and/or space. Composite samples can be obtained by combining portions of multiple grab
samples or by using specially designed automatic sampling devices.
Advantages of composite samples include reduced costs of analyzing a large number of samples,
more representative samples of heterogeneous matrices, and larger sample sizes when amounts of test
samples are limited. Disadvantages of composite samples include loss of analyte relationships in
individual samples, potential dilution of analytes below detection levels, increased potential analytical
interferences, and increased possibility of analyte interactions. In addition, use of composite samples may
reduce the number of samples analyzed below the required statistical need for specified data quality
objectives or project specific objectives.
Do not use composite samples with components or characteristics subject to significant and
unavoidable changes during storage. Analyze individual samples as soon as posible after collection and
preferably at the sampling point. Examples are dissolved gases, residual chlorine, soluble sulfde,
temperature, and pH. Changes in components such as dissolved oxygen or carbon dioxide, pH, or
 temperature may produce secondary changes in certain
alkalinity, orhardness. Inorganic constituen ts such as iron, manganese,
c
Integrated (discharge-weighted) samples: For certain purposes, the information
provided by analyzing mixtures of grab samples collected from different points needed is best
possible, using
discharge-increment (EDI) discharge-weighted methods such as equal width increment
nearly so as
procedures and
simultaneously,
(EWI) or
or as

OCCurs in a river or
stream that
varies in
equipment. An example of the need for equal
composition or total loading, use a composition across its width and depth. To
mixture of samples
integrated sampling
proportion to their relative flows. The need for representing various points in theevaluate average
cross-section,
is proposed for several separate integrated samples also may exist if in
wastewater
foct on treatability or even on composition.
streams, interaction of which may
the combined treatment
have a significant
Sampling Methods
Manual sampling: Manual sampling involves minimal
Consumingfor routine or large-scale sampling programs. Itequipment but may be unduly
costly
requires trained field technicians andandis time-
necessary for regulatory and research investigations for which critical often
cample collection techniques are appraisal field conditions and
of
containing oil and grease. essential. Manually collect certain samples, such as waters
motic sampling: Automatic
ncts may provide the meanssamplers
can eliminate human errors in
for more frequent manual sampling, can reduce
automatic sampler does not contaminate the sampling, and are used increasingly. Be sure that
omnatible with certain organic compounds that are sample. For example, plastic
components may be
aminated (e.g., from phthalate esters) by contact withsoluble in the plastic parts or that can
acordance with sampling needs.
them. Program an automatic sampler in
cSorbent sampling: Use of solid
sorbents,
renuent These methods offer advantages of rapid,particularly membrane-type disks, is becoming more
inexpensive sampling if the analytes of interest can be
adsorbed and desorbed efficiently and the water matrix is free of particulates that plug the
Sample Containers sorbent.
Containers typically are made of plastic or glass, but one material may be
other. For example, silica, preferred over the
sodium, and boron may be leached from soft glass but not
levels of same pesticides and metals may sorb onto the plastic, and trace
walls of glass containers. Thus, hard glass
containers are preferred. For samples containing organic compounds, do not use
plastic containers except
those made of fluorinated polymers such as
polytetrafluoroethylene
(PTFE).
Use glass containers for all organics analyses such as volatile organics,
semivolatile organics,
pesticides, PCBs, oil and grease. Some analytes (e.g.,bromine-containing compounds andsome pesticides.
oyuciear aromatic compounds, etc.)are light sensitive; collect them in amber-colored glass containers
Dminimize photodegradation. Container caps, typically plastic, also can be a problem. Do not use
caps
paper liners. Use foil or PTFE liners but be aware that metal liners can contaminate samples
collected for metals analysis and they may also react with the sample ifit is acidic or alkaline. Serum vials
With PTFE-lined rubber or plastic
septa are useful.
Number of Samples
Detause of variability from analytical and sampling procedures (i.e, population variability). a
single sample is insufficient to reach any reasonable desired level of confidence. If an overall standard
devi
of
ation (.e. the standard deviation offcombined sampling and analysis) is known, the required number
samples for a mobile matrix such as water may be estimated as follows.
N {(ts /U) 2
Where:
N=number of samples
 given confidence level
t Student-t statistic for a
and
S Overall standarddeviation,
To assist
Us acceptable uncertaintyuse curves. As an example, if sis 0.5mg/L, Uiss: t 0.2 mg/L, and a 95%
levelin ofcalculation,
taken.
25 to 30 samples must be
confidence level is desired, approximately
Sample Volumes
Collect a 1- sample for most physical and chemical analyses. For certain determination, larger
samples may be necessary. Do not use samples from the same container for multiple testing requirements
because matlS
(eg. organic, inorganic, radiological, bacteriological, and microscopic examinations)
collecting and handling are different for each type of test. Always collect enough sample volume in the
appropriate container in order to comply with sample handling, storage, and preservation requirements.
Sample Storage andPreservation
Neture of somplechanges: Certain cations are subject to loss by adsorption on, or ion exchange arielks
walls of glass containers. These include aluminium, cadmium, chromium, copper, iron, lead, manganeso
silver and zinc, which are best collected in a separate clean bottle and acidified with nitric acid to g nt
below 2.0te minimize precipitationand adsorption oncontainer walls.
Temperature, plH and dissolved gases (oxygen, carbon dioxide) may play major role for
characteristics of water. Many onganic compounds are sensitive to changes in pH and/or temperatura.
resating in reduced concentrations during storage. Changes in the pH-alkalinity-carbon dioxide balanca.
may cause calcium carbonate to precipitate, decreasing the values for calcium and total hardness
Iron and manganese are readily soluble in their lower oxidation states but relatively insoluble in
their higher oxidation states; therefore, these cations may
precipitate or they may dissolve from
sediment depending on the redox potential of the sample. Microbiological activity may affect the
nitrite ammonia content, phenol or BOD concentration, or the nitrate
reduction of sulfate. Residual chlorine is
reduced to chioride. Color, odor, and turbidity may increase,
and boron may be leached from the glass container.
decrease, or change in quality. Sodium, silica,
chromium.
Hexavalent chromium may be reducedto trivalent
Zero head-space is important in
radon. Avoid loss of volatile materials by
preservation of samples with volatile organic compounds and
by carefully filing the bottie sot that top ofcollecting sample in a completely filled container. Achieve this
avoid spillage or air entrapment if meniscus is above the top of the bottle rim. It is
important to
the bottie. After capping or sealingpreservatives such as HCl or ascorbic acid have already been added to
bottle, check for air bubbles by
or more air bubbies are
observed then, if practical, discard the sameinverting and gently tapping it; if one
and repeat
sampie until no air bubbles are observed (this
was filied). cannot be done if bottle containedrefilling bottle witn new
preservatives befote t
Preservation Techniques
To minimize the
potential for
sampling and analysis, keepvolatilization or
biodegradation between
freezing, Preferably pack samplessamples as cool as
in crushed or possible without
ice substitutes before cubed ice or commercial
freeze samples and mayshipment.
cause
Avoid using dry ice
because it will
may effect a pH change in glass containers to break. Dry ice
ice or a samples. Keep composite samples cool also
refrigeration system set at 49C during
samples as quickly as possible with
analysis is not possible, on arrival at thecompositing. Analyze
pH laboratory. immediate
in waterpreferably store at 4°C.
If
adjustment
below 2 by adding few samples:of The pH should be
drops nitric acid. This is brought down to
done to prevent loss of cations by adsorption, or
 the
exchange with of walls glass
orjon
manganese,
. iron, lead,
silver contalners. These include aluminum,
and zinc to
minimize precipitation and cadmium.
vNIalner
walls
preservation are relatively limited and are adsorption
wthodsoftof constituents. methods intended
Preservation
bottles refrigeration, iltration, and limited togeneral
are ly to
pB control, retard biological
opaque
rand
i n water: Changes in temperature,
freezing. chemical addition,
pH,
oxicollection.
dation-reduction
meSurement

determine
tconductamce
temperature
ovidation-reduction potential,
,turbidity alkalinity immediately after
and
may
and dissolved gasesoccur quickly
in situ: DH.
maximumholding time: Samples should not be stored over
followed. They should he prolonged periods even if
analysed for the desired parameter
holding
tìme. before the prescribed
turbidity,DO In-situ measurement
pH,EC,
24 hrs (without re
Conductivity
28 days (with frigeration)
Turbidity
refrigeration)
In-situ preferred, but can store
7 days (without upto 48 hours
Hardness acidification pH 2)
6 months (if acidified topH 2)
to

Acidity and alkalinity Within 24 hours desirable

14 days maximum
6 hours
permissible
BOD
48 hours (with proper
storage)
7days
COD
28 days with proper storage
Coliforms 6 hours
t

SOIL SAMPLING IN CONTAMINATED SITES

portant criteria
Soil excavated during boring should be sampled in such a manner that the
sample obtained is
suthiciently representative of the soil layer concerned and that the contaminant
concentrations
have not suffered evaporation or contamination caused by the packaging
or the sampling
equipment.
r Sampling at several locations in a zig-zag pattern ensures homogeneity in a relatively
homogenous soil.
Collect separate samples from sites that differ in colour, slope, drainage, cropping system and
points of contamination.
Sampling up to 15 cm depth is normally practiced. However, deepersampling is done depending
On degree of
Sampling procedure contamination and study objective.
Remove the
r
Drive the augersurface
to
debris and litters (not soil/ deposits) at the sampling spot.
the desired depth and draw the soil sample.
If
auger is using
not available, make a"V' shaped cut to a depth of 15 cm in the sampling spot
spade.
Remove thin slices of soil from top to bottom of exposed face of the 'V' shaped cut and place in a
"
clean container.
Collect atleast 10-15
samplebys repeated quartering and bring down the final volumeto 1kilogram
Mix the collected samples
 site, survev
> Label the bag providing details of the
collection, name of the sampler etc.
number, previous Crop
> Dry the samples collected from the field in shade and
sieve.
powder the sample and
sieve
grown, ate
> If the sampling is done for trace element analysis. utmost
Brass sieves should be avoided and it is better to use
collection, processing and storage of samples.
> Air drying of soils must be avoided if the
care is needed in
stainless steel or hrougT in
polyhtahnenedlingthe samole.
samples are to
ammoniacal (NH-N)nitrogen as wellas for microbial assavs. be analyzed for materials
nitrate (NO,N) and
fox
Questions
1 Which type of sampling is ideal to
characterise the
2. What are the
3. Why maximum
principal parameters to be measures for wastewater from hostel?
holding time is
Mention few important materialsrecommended? wastewater and soil
4
5. What are the related to your safety while lqualities?
6. What is catch containers
samples?
used for sampling to
determine the
sampling in water:.
7. What is different parameters?
8. composite samples?
What is different
preservation techniques used in
Class work wastewater analysis?
 FNVRON MENTAL SAMPLING
PRE.SERVATON
DATE:

laal to chacte(se tRe

Usnsteua tes
Gnab to
chaructease ttu
Asste wtes
hoste
2) keat tee
are
priencipal pasameters too bebe measwied

P", FG, Cenctuctviby ,Do, toalbidy hardnex

BOP, Coutons wate

Lempetuae, total bhnemaue euonid' sulphat

naxmus tene s hecom mended ?

peruod eve
Sample ye not be stohed oven

should the evicad Piameter

the

A) Mentton jsaompottaat mateuah helated to

Usater
G execektire heat bu
RoaoTA
 Cortuners wsed
5) khat ahe the

detamene the dejeent panamebes ?

Cotua ers wsed ko
bo detemn
e Plasttcsr and
he dtteat paramatea
tetra Fluonetayie
6)
6 What ik Catch samples
&rab [amples Con ) Catch are

sgle ked
anples clle cated at
apeatie spot at wt
u
Ove a short Ciypicaay seconda oA
mereel )Tus t
thet hephesent a snaYslot fio botk
a
area

Composcie sampes should proveda

moe
ustiak the Gonce ntia ton tte
enteest

ad /ot space peoda tum

Cera
Can be
obtaued

autematt dlevcs
8) khat ae
u0aste ater

Pheseava Eon rmetheds ale lmil to pt

Centol, chemcal adtiton , the wse amben and
bottl and

jNoRK DONÉ:
We ave coheted sotl & wrter
arourd ta
tia campus

Gneep water

Onchad Vemel wshing wa ber

2
Dí ets wa

lake Feld
3
kaidansmpath
Animal

fasttn block

Tfese clleced samples

ESTIMATION OF DISSOLVED OXYGEN (DO) AND BIOLOGICAL OXYGEN
WASTEWATER DEMAND (BOD) IN
Objective
Todetermine the purity of
water by dissolved oxygen test and to
in polluted waters by Winkler method determine the oxygen demand
Estimation of Dissolved Oxygen (DO)
The amount of oxygen dissolved in
water or the oxygen freely available in
AvYGen (D.0.) levels are considered amost water. Dissolved
desirable aquatic life. The measurement of
important indicator of water body's ability to
support
dissolved oxygen indicates the purity of water and is
imnortant for maintaining aerobiC conditions in the receiving
waters and in the aerobic treatment of
sewage and industrial waste water. The D.0. level of the
water body for the survival of the fish is 6 mg L'.
The solubility of atmospheric Oxygen in fresh
water ranges from 14.6 mg/L at 0°C to about 7.0 mg/L at
35°C under normal atmospheric pressure. Since it is
poorly soluble gas,its solubility directly varies with
the atmospheric pressure at any given
and wastewater treatment processes. The
temperature. Analysis of DO is a key test in water pollution
control
following illustrations reveal importance of DO as a
- Itis necessary to know D0 levels to
assess quality of raw water and to keep a check on streamparameter:
In wastewaters, dissolved oxygen is the pollution.
factor that determines whether the biological changes
brought out by aerobic or are
anaerobic organisms.
- DO test is the basis of BOD test which is
an important parameter to evaluate pollution potential of
wastes.
- DO is necessary for all aerobic biological
wastewater treatment processes.
-Oxygen is an important factor in corrosion. DOtest is used to
control the amount of oxygen in boiler feed
waters either by chemical or physical methods.
Sample colle ction and preservation:
Samples have to be collected in BOD bottles. Fillthe bottles without entrapment of air.
Sample
collected
in the field and brought to the lab is likely to undergo a change in DO value
because of changes
in temperature and biological reactions with time. Therefore, to obtain correct DO value, the
sample must
be fixed' immediately after collection. Fixing is done by adding 2 ml each of manganous
sulphate solution
and alkaliiodide azide reagent to the sample in the bottles. Preserve the fixed samples by keeping at 4°C
in dark.
DO estimation: Winkler method with azide modification
Principle
Oxygen present in sample rapidly oxidises the dispersed divalent manganous hydroxide to its
higher valency, which is precipitated as a brown hydrated oxide after the addition of NaOH/KOH and KI. Upon
acidification, manganese reverts to divalent state and liberates iodine from KI equivalent to the original DO
content. The liberated iodine is titrated against Na,S,0, (N/40) using starch as an indicator. The chemical
reactions involved in the method are given below:

1. MnSO, + 2KOH > Mn(0H), + K,S0. (White precipitate)

2.2 Mn(0H), + 0, +2Mn0 (OH), (Brown precipitate)
3.MnO(OH), +2H,SO), +3H,0
4. Mn(SO), + 2 KI MnS0, + K,SO, + I,
Na,S,O, + 2NaCl + 10H,0
5. 2Na,S,0,.5H,0 + I,
6.2NaN,+ H,SO, + 2HN,+ Na,SO,
7. HNO, + HN, >N, + N,0 + H,0
Apparatus
for collection of samples, c. Burettee,d. Conical fask
a. BOD bottles, capacity 300mL, b. Sampling device
 3e
1000 mL. Eiltar
MnS0,2H,0 in distilled to solution ofKI
MnSO,.4H,0or 400g to an acidified
Reagents
sulphate: Dissolve 480g starch when added
a. Manganese colour with
should not give
necessary. This solution KOH) and 150g KI (or
reagent. Dissolve 500g NaOH (or 700g
iodide-azide dissolved in 40mL
b. Alkali than saturated samples: sodium azide, NaN,
or less 10g
For saturated 1000mL. Add
solution when diluted and
acidified.
1.
distilled water and dilute to with starch
135g Nal) in give colour 480g NaOH and
solution should not 500mL distilled water. Add
distilled water. This Dissolve 10g NaN, in
samples:
2 For supersaturated produced.
dissolve the contents. hydrozoic acid fumes may
be
750g Nal and stir to because tOxic
Do not acidify this
solution alkali-iodide-azide reagent.
Cautions: equivalent to about 3mL boiling distilled
Sulphuricacid: H,SO,, conc., 1mL is of soluble starch powder in 100mL
c. of 2.0g
Prepare paste or solution
d. Starch indicator: cooland then use. Preserve by
for a few minutes,
water. Continue boiling Dissolve 24.82g Na,S,0,.5H,0 in distilled water.
thiosulphate, 0.1N:
e. Stock sodium to 1000mL.
solid NaOH or 1.5mL of 6N NaOH and dilute solution to 1000m with
adding 0.4g
thiosulphate, 0.025N: Dilute 250mL stock Na,S,O,
f Standard sodium volume. (This should
distilled water. Add preservative before making up the
freshly boiled and cooled
standard dichromate solution for each set of titrations).
be standardised with
standard potassium dichromate
Standardization of thiosulphate using distilled water
(previously dried at 103-C for 1 hour, in
Dissolve 1.2257 g potassium dichromate boiled, cooled,
250 ml in a volumetric flask. This is exactly 0.1 N solution. Place 100 ml of
and make upto the salts dissolve.
3 g of KI, 2 g NaHCO, and shake until
distilled water in a500 ml of conical flask. Add a
Sulphuric acid. Pipette 25 ml 0.1 N K,Cr,0, solution into the flask. Cover the flask with
Add 6 ml conc.
Rinse the watch glass and dilute the solution to 250 ml with
watch glass and keep it in the dark for 5 min.
the sodium thiosulphate solution in the
boiled, cooled distilled water. Titrate the liberated iodine with
add 1 ml starch indicator. Blue shade will be
burette. When the solution acquires yellowish green colour,
drop changes the colour from
obtained. Rinse the sides of the flask and continue the titration until one
thiosulphate solution.
greenish blue to light green. Calculate the normality of sodium
Procedure
Fill the BOD bottle with sample upto the rim.
Add 2ml of Manganous sulphate solution immediately followed by 2ml of alkali - iodide -azide
solution.
Stopper the bottle without entrapment of air and mix by inverting the bottle at least for about 10
minutes. The precipitate is white if the sample is devoid of oxygen, and becomes increasingly
brown with rising oxygen content.
Then add 2.0 ml of conc. H,SO,by the sides of the bottle to dissolve to the precipitate formed and
the colour of the solution turns straw yellow.
Take 100ml of sample into a conical flask
Add 2ml of starch solution and titrate the liberated iodine with the standardized sodium
thiosulphate in the burette and continue the titration by adding the thiosulphate solution in
drops tillthe disappearance of the blue colour
Repeat the experiment to obtain concordant titre values.
Calculation
1000 ml of 1Nthiosulphate = 8goxygen
TV xN x 8 x 1000
Dissolved Oxygen (mg L') =
V
 Where,
V Volume of sample in ml
TV Titre Value
N
(thiosulphate) in ml
Normality of thiosulphate used for titration
natermination of Biochemical Oxygen Demand (BOD, /BOD.)
Introduction
The Biochemical Oxygen Demand (BOD) is an empirical
oasures oXygen requirement fr aerobic standardized laboratory test
oxidation of decomposable organic matter and which
inorganic materials in water, polluted waters and certain
and incubation period. Simply, BOD is wastewater under controlled conditions of temperature
the amount of oxygen required by the
to decompose the organic matter bacteria or microorganisms
present in the waste water. It is the important
the quality of waste water and other
water body. A parameter for checking
high value of BOD indicates that there is
high
of pollutant in the water. The
maximum permissible level of BOD for drinking water is 6 mg amount
and for irrigation water is 30 mg L'. L' or ppm
The test is applied for fresh water
wastewater (domestic, industrial), polluted receiving water bodies, sources (rivers, lakes),
water) and also for finding out the level of marine water (estuaries, coastal
pollution, assimilative capacity of water body and also
performance of waste treatment plants.
Guideline BOD values for classification of raw
untreated water
Quality class Designated best use BOD value Note
Drinking water source without
A 2 or less
Conventional treatment but with
Could cause problems in
chlorination
treatment, larger Cl, demand and
B
Drinking water source with residual taste/odour problem.
3 or less
Conventional treatment
Principle
BOD is evaluated by measuring oxygen concentration in samples idometrically before and after
incubation in the dark at 20C for 5 days. Preliminary dilution and aeration of sample are usually
necessary to ensure that not all the oxygen is consumed during incubation. Samples absorbing more than
6mg/l of dissolved oxygen should be diluted with dilution water. Generally sewage is used as astandard
seed material. Polyseed (Specialized bacterial cultures) is also used as seeding material.
Apparatus
a. BOD bottles, capacity 300mL, b. Sampling device for collection of samples, c. Burettee, d. Conical flask
Reagents:
(A) Allthe reagents used for D.0 test.
(B) Water for dilution
Take 2 litre distilled water in 3 litres bottle. Shake this partially filled bottle for about ten
minutes so that the distilled water gets saturated with atmospheric oxygen or using aerated, the distilled
water can be aerated for 1 or 2 days. Then add to this 2 ml of phosphate buffer (pH 7.0) solution, 2 ml of
MgSO, solution, 2 ml CaCl, solution and 2 ml of ferric chloride solution.
K,HPO, and 1.7 g NH,Cl in 500
(a) Phosphate buffer solution: Dissolve 8.5 g of KH,PO, 21.75 g
ml distilled water. Dilute this solution to 1 litre.
water and dilute the solution to 1 litre.
(b) Ferricchloride: Dissolve 0.25 g FeCl,.6H,0 in
of distilled water.
(c) Calcium chloride: Dissolve 0.25 g CaCl, in 1 litre water.
Dissolve 22.5 g MgSO.7H,0 in 1litre of distilled
(d) Magnesium sulphate solution:
 Procedure dilution: BOD determination.
Sample with very low
determination without used as such for
BOD
is fairly clean,
it can be require seeding with a small known
Ifthe sample stabilized in 5 days. These samples BOD value ic
bacterial content may not
get
done, a seed correction in the measure seedino
is
sewagewater. When it sewage water used or
volume of domestic measuring the BOD value of
is determined by
The value for correction upto the rim.
Filltwo BOD bottles
stopper these bottles.
After 15 min., tightly the bottles.
no air bubbles inside
Make sure that there are measurement.
for initial dissolved oxygen
Use the sample in one bottle
incubator at 20°C for 5 days.
Place the other bottle in an
oxygen in the incubated bottle.
After 5 days, determine the dissolved
BOD determinationwith dilution
Determine the D0 of the diluted sample
This method is used when the BOD value exceeds 5.
bottle for 5 days at 20°C and determine the D0
using one of the bottles immediately. Incubate the other
Calculation
a. Sample without dilution
(BOD,/BOD,) = DO, - DO, mg L
b. Sample with dilution

BOD, /BOD, (mg/) =(DO,- DO,- BC) XDF

where,
DO, is the initial dissolved oxygen content in mg/1.
DO, is the dissolved oxygen content after incubation for5 days.
BCis the difference between the dissolved oxygen contents of the blank on initial day and after 5
days of incubation (Blank correction).
c. Sample with dilution and seeding BOD, /BOD, =BOD of sample - BOD of seeding sample
(sewage)
Where,
DO, DO of diluted water sample before
incubation
DO, DOof diluted water sample after incubation
Note:
1. Ifyou use seeding, subtract the BOD
of seeding sample with total BOD.
2. For reliable results
following conditions are essential:
a. Minimum depletion of DO of
2mg/L
b. Minimum residual DO of
1mg/L at the end of test period
c. 5days 20°C or 3days 27°C
B0D value of
Dilution of sample required for various 5mg/L can be measured directly without dilution
Expected BOD of sample (mg L) ranges of BOD
0-6 Volume of sample (ml/) of mixture
1000
Dilution factor
4-12
1
10-30 500
2
20-60 200
5
40-120 100
10
100-300 50
20
200-600 20
400-1200 10 50
1000-3000 100
2000-6000 200
More than 6000 500
0.5 1000
2000
 (OR)
Generalguideliness for dilution range are as follows (sample content in percentage) :
0.1%to 1% Strong trade waste
1%to5% : Raw or settled sewage
59%to259% Treated effluent
25%to 100% River water
Technological advancement
Readyto use DO meters and DO test kits have been developed and are in use for field studies.

100.5%

DOmeter DO pen DO probe for bottom sampling

 A. No: FSMAtton! Do

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DATE:

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